Petroleum derived fuels have been the primary source of energy for over a hundred years. Petroleum, however, has formed over millions of years in nature and is not a renewable source of energy. A significant amount of research in alternative fuels has been ongoing for decades. Within this field, ethanol has been studied intensively as a gasoline substitute and the use of ethanol as transportation fuel has been increasing recently (Gray et al., Curr Opin Chem Biol 2006, 10:141). However, the efficiency of ethanol as a fuel is still in debate (Pimentel, Natural Resources Research 2005, 14:65; Farrell et al., Science 2006, 311:506). There is interest to design several potential alternative fuel molecules other than ethanol, which can be produced biosynthetically, and to develop the biosynthetic pathways for enhanced production of the target fuel molecules using synthetic biology.
Currently, gasoline and diesel fuels are the two major transportation fuels. Gasoline is a complex mixture of hydrocarbons and additives for improving fuel performance. The carbon number of hydrocarbons in gasoline varies from 4 to 12, with branched alkanes, cyclic alkanes and aromatics being the most abundant. Diesel fuel is a mixture of many different hydrocarbons with the carbon numbers ranging from 9 to 23 with an average of 16. Usually n-alkanes and oxygenates in diesel fuel tend to increase the octane number, while branched or unsaturated hydrocarbons lower this value. Biodiesel has been of interest recently as a promising alternative fuel due to its renewability and environmental benefits. Biodiesel is a mixture of monoalkyl esters of long chain fatty acids derived from vegetable oils or animal fats. It is typically produced by acid or base-catalyzed transesterification of glycerin with methanol. The introduction of ester functionality in biodiesel improved the fuel properties, such as a higher octane number and an increased lubricating effect. Recently, enzymatic processes for fatty acid transesterification have been reported for the production of biodiesel (Kalscheuer et al., Microbiol. 2006, 152:2529-2536).
This present invention involves the biosynthesis of two hydrocarbons: isoprenoids and fatty acids. Isoprenoids are compounds derived from the five-carbon molecule, isopentenyl pyrophosphate. Investigators have identified over 29,000 individual isoprenoid compounds, with new ones continuously being discovered. Isoprenoids are often isolated from natural products, such as plants and microorganisms, which use isopentenyl pyrophosphate as a basic building block to form relatively complex structures. Vital to living organisms, isoprenoids serve to maintain cellular fluidity and electron transport, as well as function as natural pesticides, to name just a few of their roles in vivo. Furthermore, the pharmaceutical and chemical communities use isoprenoids as pharmaceuticals, nutriceuticals, flavoring agents, and agricultural pest control agents. Given their importance in biological systems and usefulness in a broad range of applications, isoprenoids have been the focus of much attention by scientists.
Conventional means for producing isoprenoids include extraction from biological materials (e.g., plants, microbes, and animals) and partial or total organic synthesis in the laboratory. Such means, however, have generally unsatisfactory as they involve the use of toxic solvents and provide a low yield of the desired isoprenoid. Recently, researchers have looked to the biosynthetic production of isoprenoids. U.S. Pat. No. 6,291,745 describes the production of limonene and other metabolites in plants. U.S. Pat. No. 6,190,895 describes nucleic acid sequences that code for the expression of 1-deoxyxylulose-5-phosphate synthase, an enzyme used in one biological pathway for the synthesis of isopentenyl pyrophosphate. U.S. Pat. No. 7,172,886 describes the cloning of genes for a mevalonate-isoprenoid pathway and synthesizing an isoprenoid or an isoprenoid precursor via the mevalonate pathway in a host cell. U.S. Pat. No. 7,183,089 describes a method for enhancing production of isoprenoid compounds in a host cell by modulating the level of hydroxymethylglutaryl-CoA (HMG-CoA) in the cell, such that the level of HMG-CoA is not toxic to the cell and does not substantially inhibit cell growth.
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